Growth differentiation factor-11

ABSTRACT

Growth differentiation factor-11 (GDF-11) is disclosed along with its polynucleotide sequence and amino acid sequence. The invention provides a method for identifying a compound that affects GDF-11 activity or gene expression.

This application is a continuation of U.S. Ser. No. 09/871,604, filedMay 31, 2001 (now abandoned), which is a continuation of U.S. Ser. No.09/123,929, filed Jul. 28, 1998 (now abandoned), which is acontinuation-in-part application of U.S. Ser. No. 09/019,901, filed Feb.6, 1998 (now abandoned), which is a continuation-in-part of U.S. Ser.No. 08/795,671, filed Feb. 6, 1997 (now U.S. Pat. No. 6,008,434), and isa continuation-in-part of U.S. Ser. No. 08/706,958, filed Sep. 3, 1996(now abandoned), which is a continuation of U.S. Ser. No. 08/272,763,filed Jul. 8, 1994 (now abandoned), the entire content of each of whichis incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to growth factors and specifically to anew member of the transforming growth factor beta (TGF-β) superfamily,which is denoted, growth differentiation factor-11 (GDF-11) and methodsof use for modulating muscle cell, bone, kidney and adipose tissuegrowth.

2. Description of Related Art

The transforming growth factor β (TGF-β) superfamily encompasses a groupof structurally-related proteins which affect a wide range ofdifferentiation processes during embryonic development. The familyincludes, Mullerian inhibiting substance (MIS), which is required fornormal male sex development (Behringer, et al., Nature, 345:167, 1990),Drosophila decapentaplegic (DPP) gene product, which is required fordorsal-ventral axis formation and morphogenesis of the imaginal disks(Padgett, et al., Nature, 325:81-84, 1987), the Xenopus Vg-1 geneproduct, which localizes to the vegetal pole of eggs (Weeks, et al.,Cell, 51:861-867, 1987), the activins (Mason, et al., Biochem, Biophys.Res. Comm., 135:957-964, 1986), which can induce the formation ofmesoderm and anterior structures in Xenopus embryos (Thomsen, et al.,Cell, 63:485, 1990), and the bone morphogenetic proteins (BMPs,osteogenin, OP-1) which can induce de novo cartilage and bone formation(Sampath, et al., J. Biol. Chem., 265:13198, 1990). The TGF-βs caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, hematopoiesis, and epithelialcell differentiation (for review, see Massague, Cell 49:437, 1987).

The proteins of the TGF-β family are initially synthesized as a largeprecursor protein which subsequently undergoes proteolytic cleavage at acluster of basic residues approximately 110-140 amino acids from theC-terminus. The C-terminal regions, or mature regions, of the proteinsare all structurally related and the different family members can beclassified into distinct subgroups based on the extent of theirhomology. Although the homologies within particular subgroups range from70% to 90% amino acid sequence identity, the homologies betweensubgroups are significantly lower, generally ranging from only 20% to50%. In each case, the active species appears to be a disulfide-linkeddimer of C-terminal fragments. Studies have shown that when thepro-region of a member of the TGF-β family is coexpressed with a matureregion of another member of the TGF-β family, intracellular dimerizationand secretion of biologically active homodimers occur (Gray, A. et al.,Science, 247:1328, 1990). Additional studies by Hammonds, et al.,(Molec. Endocrinol. 5:1-49, 1991) showed that the use of the BMP-2pro-region combined with the BMP-4 mature region led to dramaticallyimproved expression of mature BMP-4. For most of the family members thathave been studied, the homodimeric species has been found to bebiologically active, but for other family members, like the inhibins(Ling, et al., Nature, 321:779, 1986) and the TGF-βs (Cheifetz, et al.,Cell, 48:409, 1987), heterodimers have also been detected, and theseappear to have different biological properties than the respectivehomodimers.

In addition, it is desirable to produce livestock and game animals, suchas cows, sheep, pigs, chicken and turkey, fish which are relatively highin musculature and protein, and low in fat content. Many drug and dietregimens exist which may help increase muscle and protein content andlower undesirably high fat and/or cholesterol levels, but such treatmentis generally administered after the fact, and is begun only aftersignificant damage has occurred to the vasculature. Accordingly, itwould be desirable to produce animals which are genetically predisposedto having higher muscle content, without any ancillary increase in fatlevels.

The food industry has put much effort into increasing the amount ofmuscle and protein in foodstuffs. This quest is relatively simple in themanufacture of synthetic foodstuffs, but has been met with limitedsuccess in the preparation of animal foodstuffs. Attempts have beenmade, for example, to lower cholesterol levels in beef and poultryproducts by including cholesterol-lowering drugs in animal feed (seee.g. Elkin and Rogler, J. Agric. Food Chem. 1990, 38:1635-1641).However, there remains a need for more effective methods of increasingmuscle and reducing fat and cholesterol levels in animal food products.

SUMMARY OF THE INVENTION

The present invention provides a cell growth and differentiation factor,GDF-11, a polynucleotide sequence which encodes the factor, andantibodies which are immunoreactive with the factor. This factor appearsto relate to various cell proliferative disorders, especially thoseinvolving muscle, nerve, bone, kidney and adipose tissue.

In one embodiment, the invention provides a method for detecting a cellproliferative disorder of muscle, nerve, bone, kidney or fat origin andwhich is associated with GDF-11. In another embodiment, the inventionprovides a method for treating a cell proliferative disorder bysuppressing or enhancing GDF-11 activity.

In another embodiment, the subject invention provides non-humantransgenic animals which are useful as a source of food products withhigh muscle, bone and protein content, and reduced fat and cholesterolcontent. The animals have been altered chromosomally in their germ cellsand somatic cells so that the production of GDF-11 is produced inreduced amounts, or is completely disrupted, resulting in animals withdecreased levels of GDF-11 in their system and higher than normal levelsof muscle tissue and bone tissue, such as ribs, preferably withoutincreased fat and/or cholesterol levels. Accordingly, the presentinvention also includes food products provided by the animals. Such foodproducts have increased nutritional value because of the increase inmuscle tissue and bone tissue to which the muscle attaches. Thetransgenic non-human animals of the invention include bovine, porcine,ovine and avian animals, for example.

The subject invention also provides a method of producing animal foodproducts having increased muscle content. The method includes modifyingthe genetic makeup of the germ cells of a pronuclear embryo of theanimal, implanting the embryo into the oviduct of a pseudopregnantfemale thereby allowing the embryo to mature to full term progeny,testing the progeny for presence of the transgene to identifytransgene-positive progeny, cross-breeding transgene-positive progeny toobtain further transgene-positive progeny and processing the progeny toobtain foodstuff. The modification of the germ cell comprises alteringthe genetic composition so as to disrupt or reduce the expression of thenaturally occurring gene encoding for production of GDF-11 protein. In aparticular embodiment, the transgene comprises antisense polynucleotidesequences to the GDF-11 protein. Alternatively, the transgene maycomprise a non-functional sequence which replaces or intervenes in thenative GDF-11 gene.

The subject invention also provides a method of producing animal foodproducts having increased bone content. The method includes modifyingthe genetic makeup of the germ cells of a pronuclear embryo of theanimal, implanting the embryo into the oviduct of a pseudopregnantfemale thereby allowing the embryo to mature to full term progeny,testing the progeny for presence of the transgene to identifytransgene-positive progeny, cross-breeding transgene-positive progeny toobtain further transgene-positive progeny and processing the progeny toobtain foodstuff. The modification of the germ cell comprises alteringthe genetic composition so as to disrupt or reduce the expression of thenaturally occurring gene encoding for production of GDF-11 protein. In aparticular embodiment, the transgene comprises antisense polynucleotidesequences to the GDF-11 protein. Alternatively, the transgene maycomprise a non-functional sequence which replaces or intervenes in thenative GDF-11 gene.

The subject invention also provides a method of producing avian foodproducts having improved muscle and/or bone content. The method includesmodifying the genetic makeup of the germ cells of a pronuclear embryo ofthe avian animal, implanting the embryo into the oviduct of apseudopregnant female into an embryo of a chicken, culturing the embryounder conditions whereby progeny are hatched, testing the progeny forpresence of the genetic alteration to identify transgene-positiveprogeny, cross-breeding transgene-positive progeny and processing theprogeny to obtain foodstuff.

The invention also provides a method for treating a muscle, bone, kidneyor adipose tissue disorder in a subject. The method includesadministering a therapeutically effective amount of a GDF-11 agent tothe subject, thereby affecting growth of muscle, bone, kidney or adiposetissue. The GDF-11 agent may include an antibody, a GDF-11 antisensemolecule or a dominant negative polypeptide, for example. In one aspect,a method for inhibiting the growth regulating actions of GDF-11 bycontacting an anti-GDF-11 monoclonal antibody, a GDF-11 antisensemolecule or a dominant negative polypeptide (or polynucleotide encodinga dominant negative polypeptide) with fetal or adult muscle cells orprogenitor cells is included. These agents can be administered to apatient suffering from a disorder such as muscle wasting disease,neuromuscular disorder, muscle atrophy, obesity or other adipocyte celldisorders, and aging, for example. In another aspect of the invention,the agent may be an agonist of GDF-11 activity.

The invention also provides a method for identifying a compound thataffects GDF-11 activity or gene expression including incubating thecompound with GDF-11 polypeptide, or with a recombinant cell expressingGDF-11 under conditions sufficient to allow the compounds to interactand determining the effect of the compound on GDF-11 activity orexpression.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the nucleotide and predicted amino acid sequencesof murine (FIG. 1A; SEQ ID NOS:3 and 4, respectively) and human (FIG.1B; SEQ ID NOS:1 and 2, respectively) GDF-11. The putative proteolyticprocessing sites are shown by the shaded boxes. In the human sequence,the potential N-linked glycosylation signal is shown by the open box,and the consensus polyadenylation signal is underlined; the poly A tailis not shown.

FIGS. 2A and 2B show northern blots of RNA prepared from adult (FIG. 2A)or fetal and neonatal (FIG. 2B) tissues probed with a murine GDF-11probe.

FIG. 3 shows amino acid homologies among different members of the TGF-βsuperfamily. Numbers represent percent amino acid identities betweeneach pair calculated from the first conserved cysteine to theC-terminus. Boxes represent homologies among highly-related memberswithin particular subgroups.

FIG. 4A shows an alignment of the predicted amino acid sequences ofhuman GDF-11 (top lines; SEQ ID NO:2) with human GDF-8 (bottom lines;SEQ ID NO:5). Vertical lines indicate identities. Dots represent gapsintroduced in order to maximize the alignment. Numbers represent aminoacid positions relative to the N-terminus. The putative proteolyticprocessing sites are shown by the open box. The conserved cysteineresidues on the C-terminal region are shown by the shaded boxes.

FIG. 4B shows the predicted amino acid sequences of murine (SEQ ID NO:4)and human (SEQ ID NO:2) GDF-11 aligned with murine (McPherron et al.,1997; SEQ ID NO:6) and human (McPherron and Lee, 1997; SEQ ID NO:5)myostatin (MSTN). Shaded boxes represent amino acid homology with themurine and human GDF-11 sequences. Amino acids are numbered relative tothe human GDF-11 sequence. The predicted proteolytic processing sitesare located at amino acids 295-298.

FIG. 5 shows the expression of GDF-11 in mammalian cells. Conditionedmedium prepared from Chinese hamster ovary cells transfected with ahybrid GDF-8/GDF-11 gene (see text) cloned into the MSXND expressionvector in either the antisense (lane 1) or sense (lane 2) orientationwas dialyzed, lyophilized, and subjected to western blot analysis usingantibodies directed against the C-terminal portion of GDF-8 protein.Arrows at right indicate the putative unprocessed (pro-GDF-8/GDF-11) orprocessed GDF-11 proteins. Numbers at left indicate mobilities ofmolecular weight standards.

FIG. 6 shows the chromosomal mapping of human GDF-11. DNA samplesprepared from human/rodent somatic cell lines were subjected to PCR,electrophoresed on agarose gels, blotted, and probed. The humanchromosome contained in each of the hybrid cell lines is identified atthe top of each of the first 24 lanes (1-22, X, and Y). In the lanesdesignated CHO, M, and H, the starting DNA template was total genomicDNA from hamster, mouse, and human sources, respectively. In the lanemarked B1, no template DNA was used. Numbers at left indicate themobilities of DNA standards.

FIGS. 7A to 7C show the FISH localization of GDF-11. Metaphasechromosomes derived from peripheral blood lymphocytes were hybridizedwith digoxigenin-labeled human GDF-11 probe (FIG. 7A) or a mixture ofhuman GDF-11 genomic and chromosome 12-specific centromere probes (FIG.7B) and analyzed as described in the text. A schematic showing thelocation of GDF-11 at position 12q13 is shown in FIG. 7C.

FIG. 8 shows a genomic Southern analysis of DNA isolated from differentspecies.

FIGS. 9A and 9B show the construction of GDF-11 null mice by homologoustargeting.

FIG. 9A is a map of the GDF-11 locus (top line) and targeting construct(second line). The black and stippled boxes represent coding sequencesfor the pro-and C-terminal regions, respectively. The targetingconstruct contains a total of 11 kb of homology with the GDF-11 gene. Aprobe derived from the region upstream of the 3′ homology fragment anddownstream of the first EcoRI site shown hybridizes to a 6.5 kb EcoRIfragment in the GDF-11 gene and a 4.8 kb fragment in a homologouslytargeted gene. Abbreviations: X, XbaI; E, EcoRI.

FIG. 9B shows a genomic Southern blot of DNA prepared from F1heterozygous mutant mice (lanes 1 and 2) and offspring derived from amating of these mice (lanes 3-12).

FIG. 10 shows kidney abnormalities in GDF-11 knockout mice. Kidneys ofnewborn animals were examined and classified according to the number ofnormal sized or small kidneys as shown at the top. Numbers in the tableindicate number of animals falling into each classification according togenotype.

FIGS. 11A to 11J show homeotic transformations in GDF-11 mutant mice.FIG. 11A shows newborn pups with missing (first and second from left)and normal looking tails. FIGS. 11B to 11J show skeleton preparationsfor newborn wild-type (FIGS. 11B, 11E and 11H), heterozygous (FIGS. 11C,11F, and 11I) and homozygous (FIGS. 11D, 11G and 11J) mutant mice. Wholeskeleton preparations (FIGS. 11B to 11D), vertebral columns (FIGS. 11Eto 11G), vertebrosternal ribs (FIGS. 11H to 11J) showing transformationsand defects in homozygous and heterozygous mutant mice. Numbers indicatethoracic segments.

FIG. 12 is a table summarizing the anterior transformations inwild-type, heterozygous and homozygous GDF-11 mice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a growth and differentiation factor,GDF-11, and a polynucleotide sequence encoding GDF-11. GDF-11 isexpressed at highest levels in muscle, brain, uterus, spleen, and thymusand at lower levels in other tissues.

The TGF-β superfamily consists of multifunctional polypeptides thatcontrol proliferation, differentiation, and other functions in many celltypes. Many of the peptides have regulatory, both positive and negative,effects on other peptide growth factors. The structural homology betweenthe GDF-11 protein of this invention and the members of the TGF-βfamily, indicates that GDF-11 is a new member of the family of growthand differentiation factors. Based on the known activities of many ofthe other members, it can be expected that GDF-11 will also possessbiological activities that will make it useful as a diagnostic andtherapeutic reagent.

Certain members of this superfamily have expression patterns or possessactivities that relate to the function of the nervous system. Forexample, one family member, namely GDNF, has been shown to be a potentneurotrophic factor that can promote the survival of dopaminergicneurons (Lin, et al., Science, 260:1130). Another family member, namelydorsalin-1, is capable of promoting the differentiation of neural crestcells (Basler, et al., Cell, 73:687, 1993). The inhibins and activinshave been shown to be expressed in the brain (Meunier, et al., Proc.Nat'l. Acad. Sci., USA, 85:247, 1988; Sawchenko, et al., Nature,334:615, 1988), and activin has been shown to be capable of functioningas a nerve cell survival molecule (Schubert, et al., Nature, 344:868,1990). Another family member, namely GDF-1, is nervous system-specificin its expression pattern (Lee, Proc. Nat'l. Acad. Sci., USA, 88:4250,1991), and certain other family members, such as Vgr-1 (Lyons, et al.,Proc. Nat'l. Acad. Sci., USA, 86:4554, 1989; Jones, et al., Development,111:581, 1991), OP-1 (Ozkaynak, et al., J. Biol. Chem., 267:25220,1992), and BMP-4 (Jones, et al., Development, 111:531, 1991), are alsoknown to be expressed in the nervous system. The expression of GDF-11 inbrain and muscle suggests that GDF-11 may also possess activities thatrelate to the function of the nervous system. In particular, it isknown, for example, that skeletal muscle produces a factor or factorsthat promote the survival of motor neurons (Brown, Trends Neurosci.,7:10, 1984). The known neurotrophic activities of other members of thisfamily and the expression of GDF-11 in muscle suggest that one activityof GDF-11 may be as a trophic factor for motor neurons; indeed, GDF-11is highly related to GDF-8, which is virtually muscle-specific in itsexpression pattern. Alternatively, GDF-11 may have neurotrophicactivities for other neuronal populations. Hence, GDF-11 may have invitro and in vivo applications in the treatment of neurodegenerativediseases, such as amyotrophic lateral sclerosis, or in maintaining cellsor tissues in culture prior to transplantation.

GDF-11 may also have applications in treating disease processesinvolving the musculoskeletal system, such as in musculodegenerativediseases, osteoporosis or in tissue repair due to trauma. In thisregard, many other members of the TGF-β family are also importantmediators of tissue repair. TGF-β has been shown to have marked effectson the formation of collagen and to cause a striking angiogenic responsein the newborn mouse (Roberts, et al., Proc. Natl. Acad. Sci., USA83:4167, 1986). TGF-β has also been shown to inhibit the differentiationof myoblasts in culture (Massague, et al., Proc. Natl. Acad. Sci., USA83:8206, 1986). Moreover, because myoblast cells may be used as avehicle for delivering genes to muscle for gene therapy, the propertiesof GDF-11 could be exploited for maintaining cells prior totransplantation or for enhancing the efficiency of the fusion process.GDF-11 may also have applications in treating disease processesinvolving the kidney or in kidney repair due to trauma.

GDF-11 may also have applications in the treatment of immunologic,disorders. In particular, TGF-β has been shown to have a wide range ofimmunoregulatory activities, including potent suppressive effects on Band T cell proliferation and function (for review, see Palladino, etal., Ann. N.Y. Acad. Sci., 593:181, 1990). The expression of GDF-11 inspleen and thymus suggests that GDF-11 may possess similar activitiesand therefore, may be used as an anti-inflammatory agent or as atreatment for disorders related to abnormal proliferation or function oflymphocytes.

The animals contemplated for use in the practice of the subjectinvention are those animals generally regarded as useful for theprocessing of food stuffs, i.e. avian such as meat bred and egg layingchicken and turkey, ovine such as lamb, bovine such as beef cattle andmilk cows, piscine and porcine. For purposes of the subject invention,these animals are referred to as “transgenic” when such animal has had aheterologous DNA sequence, or one or more additional DNA sequencesnormally endogenous to the animal (collectively referred to herein as“transgenes”) chromosomally integrated into the germ cells of theanimal. The transgenic animal (including its progeny) will also have thetransgene fortuitously integrated into the chromosomes of somatic cells.

The TGF-β superfamily consists of multifunctional polypeptides thatcontrol proliferation, differentiation, and other functions in many celltypes. Many of the peptides have regulatory, both positive and negative,effects on other peptide growth factors. The structural homology betweenthe GDF-11 protein of this invention and the members of the TGF-βfamily, indicates that GDF-11 is a new member of the family of growthand differentiation factors. Based on the known activities of many ofthe other members, it can be expected that GDF-11 will also possessbiological activities that will make it useful as a diagnostic andtherapeutic reagent.

In particular, certain members of this superfamily have expressionpatterns or possess activities that relate to the function of thenervous system. For example, the inhibins and activins have been shownto be expressed in the brain (Meunier, et al., Proc. Natl. Acad. Sci.,USA, 85:247, 1988; Sawchenko, et al., Nature, 334:615, 1988), andactivin has been shown to be capable of functioning as a nerve cellsurvival molecule (Schubert, et al., Nature, 344:868, 1990). Anotherfamily member, namely, GDF-1, is nervous system-specific in itsexpression pattern (Lee, S. J., Proc. Natl. Acad. Sci., USA, 88:4250,1991), and certain other family members, such as Vgr-1 (Lyons, et al.,Proc. Natl. Acad. Sci., USA, 86:4554, 1989; Jones, et al., Development,111:531, 1991), OP-1 (Ozkaynak, et al., J. Biol. Chem., 267:25220,1992), and BMP-4 (Jones, et al., Development, 111:531, 1991), are alsoknown to be expressed in the nervous system. Because it is known thatskeletal muscle produces a factor or factors that promote the survivalof motor neurons (Brown, Trends Neurosci., 7:10, 1984), the expressionof GDF-11 in muscle suggests that one activity of GDF-11 may be as atrophic factor for neurons. In this regard, GDF-11 may have applicationsin the treatment of neurodegenerative diseases, such as amyotrophiclateral sclerosis or muscular dystrophy, or in maintaining cells ortissues in culture prior to transplantation.

The expression of GDF-11 in adipose tissue also raises the possibilityof applications for GDF-11 in the treatment of obesity or of disordersrelated to abnormal proliferation of adipocytes. In this regard, TGF-βhas been shown to be a potent inhibitor of adipocyte differentiation invitro (Ignotz and Massague, Proc. Natl. Acad. Sci., USA 82:8530, 1985).

Polypeptides, Polynucleotides, Vectors and Host Cells

The invention provides substantially pure GDF-11 polypeptide andisolated polynucleotides that encode GDF-11. The term “substantiallypure” as used herein refers to GDF-11 which is substantially free ofother proteins, lipids, carbohydrates or other materials with which itis naturally associated. One skilled in the art can purify GDF-11 usingstandard techniques for protein purification. The substantially purepolypeptide will yield a single major band on a non-reducingpolyacrylamide gel. The purity of the GDF-11 polypeptide can also bedetermined by amino-terminal amino acid sequence analysis. GDF-11polypeptide includes functional fragments of the polypeptide, as long asthe activity of GDF-11 remains. Smaller peptides containing thebiological activity of GDF-11 are included in the invention.

The invention provides polynucleotides encoding the GDF-11 protein.These polynucleotides include DNA, cDNA and RNA sequences which encodeGDF-11. It is understood that all polynucleotides encoding all or aportion of GDF-11 are also included herein, as long as they encode apolypeptide with GDF-11 activity. Such polynucleotides include naturallyoccurring, synthetic, and intentionally manipulated polynucleotides. Forexample, GDF-11 polynucleotide may be subjected to site-directedmutagenesis. The polynucleotide sequence for GDF-11 also includesantisense sequences. The polynucleotides of the invention includesequences that are degenerate as a result of the genetic code. There are20 natural amino acids, most of which are specified by more than onecodon. Therefore, all degenerate nucleotide sequences are included inthe invention as long as the amino acid sequence of GDF-11 polypeptideencoded by the nucleotide sequence is functionally unchanged.

Specifically disclosed herein is a DNA sequence containing the humanGDF-11 gene. The sequence contains an open reading frame encoding apolypeptide 407 amino acids in length. The sequence contains a putativeRXXR (SEQ ID NO:12) proteolytic cleavage site at amino acids 295-298.Cleavage of the precursor at this site would generate an activeC-terminal fragment 109 amino acids in length with a predicted molecularweight of approximately 12,500 kDa. Also disclosed herein is a partialmurine genomic sequence. Preferably, the human GDF-11 nucleotidesequence is SEQ ID NO:1 and the mouse nucleotide sequence is SEQ IDNO:3.

The polynucleotide encoding GDF-11 includes SEQ ID NO:1 and 3, as wellas nucleic acid sequences complementary to SEQ ID NOS:1 and 3. Acomplementary sequence may include an antisense nucleotide. When thesequence is RNA, the deoxyribonucleotides A, G, C, and T of SEQ ID NO:1and 3 are replaced by ribonucleotides A, G, C, and U, respectively. Alsoincluded in the invention are fragments of the above-described nucleicacid sequences that are at least 15 bases in length, which is sufficientto permit the fragment to selectively hybridize to DNA that encodes theprotein of SEQ ID NO:1 or 3 under physiological conditions (e.g., understringent conditions).

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

An example of progressively higher stringency conditions is as follows:2×SSC/0.1% SDS at about room temperature (hybridization conditions);0.2×SSC/0.1% SDS at about room temperature (low stringency conditions);0.2×SSC/0.1% SDS at about 42° C. (moderate stringency conditions); and0.1×SSC at about 68° C. (high stringency conditions). Washing can becarried out using only one of these conditions, e.g., high stringencyconditions, or each of the conditions can be used, e.g., for 10-15minutes each, in the order listed above, repeating any or all of thesteps listed. However, as mentioned above, optimal conditions will vary,depending on the particular hybridization reaction involved, and can bedetermined empirically.

The C-terminal region of GDF-11 following the putative proteolyticprocessing site shows significant homology to the known members of theTGF-β superfamily. The GDF-11 sequence contains most of the residuesthat are highly conserved in other family members (see FIG. 1). Like theTGF-βs and inhibin βs, GDF-11 contains an extra pair of cysteineresidues in addition to the 7 cysteines found in virtually all otherfamily members. Among the known family members, GDF-11 is mosthomologous to GDF-8 (92% sequence identity; see FIG. 3).

Minor modifications of the recombinant GDF-11 primary amino acidsequence may result in proteins which have substantially equivalentactivity as compared to the GDF-11 polypeptide described herein. Suchmodifications may be deliberate, as by site-directed mutagenesis, or maybe spontaneous. All of the polypeptides produced by these modificationsare included herein as long as the biological activity of GDF-11 stillexists. Further, deletion of one or more amino acids can also result ina modification of the structure of the resultant molecule withoutsignificantly altering its biological activity. This can lead to thedevelopment of a smaller active molecule which would have broaderutility. For example, one can remove amino or carboxy terminal aminoacids which are not required for GDF-11 biological activity.

The nucleotide sequence encoding the GDF-11 polypeptide of the inventionincludes a disclosed sequence (SEQ ID NOS:2 and 4) and conservativevariations thereof. The term “conservative variation” as used hereindenotes the replacement of an amino acid residue by another,biologically similar residue. Examples of conservative variationsinclude the substitution of one hydrophobic residue such as isoleucine,valine, leucine or methionine for another, or the substitution of onepolar residue for another, such as the substitution of arginine forlysine, glutamic for aspartic acid, or glutamine for asparagine, and thelike. The term “conservative variation” also includes the use of asubstituted amino acid in place of an unsubstituted parent amino acidprovided that antibodies raised to the substituted polypeptide alsoimmunoreact with the unsubstituted polypeptide.

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization techniques whichare well known in the art. These include, but are not limited to: 1)hybridization of genomic or cDNA libraries with probes to detecthomologous nucleotide sequences, 2) polymerase chain reaction (PCR) ongenomic DNA or cDNA using primers capable of annealing to the DNAsequence of interest, and 3) antibody screening of expression librariesto detect cloned DNA fragments with shared structural features.

Preferably the GDF-11 polynucleotide of the invention is derived from amammalian organism, and most preferably from mouse, rat, cow, pig, orhuman. GDF-11 polynucleotides from chicken, fish and other species arealso included herein. Screening procedures which rely on nucleic acidhybridization make it possible to isolate any gene sequence from anyorganism, provided the appropriate probe is available. Oligonucleotideprobes, which correspond to a part of the sequence encoding the proteinin question, can be synthesized chemically. This requires that short,oligopeptide stretches of amino acid sequence must be known. The DNAsequence encoding the protein can be deduced from the genetic code,however, the degeneracy of the code must be taken into account. It ispossible to perform a mixed addition reaction when the sequence isdegenerate. This includes a heterogeneous mixture of denatureddouble-stranded DNA. For such screening, hybridization is preferablyperformed on either single-stranded DNA or denatured double-strandedDNA. Hybridization is particularly useful in the detection of cDNAclones derived from sources where an extremely low amount of mRNAsequences relating to the polypeptide of interest are present. In otherwords, by using stringent hybridization conditions directed to avoidnon-specific binding, it is possible, for example, to allow theautoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Wallace, et al., Nucl. Acid Res.9:879, 1981).

The development of specific DNA sequences encoding GDF-11 can also beobtained by: 1) isolation of double-stranded DNA sequences from thegenomic DNA; 2) chemical manufacture of a DNA sequence to provide thenecessary codons for the polypeptide of interest; and 3) in vitrosynthesis of a double-stranded DNA sequence by reverse transcription ofmRNA isolated from a eukaryotic donor cell. In the latter case, adouble-stranded DNA complement of mRNA is eventually formed which isgenerally referred to as cDNA.

Of the three above-noted methods for developing specific DNA sequencesfor use in recombinant procedures, the isolation of genomic DNA isolatesis the least common. This is especially true when it is desirable toobtain the microbial expression of mammalian polypeptides due to thepresence of introns.

The synthesis of DNA sequences is frequently the method of choice whenthe entire sequence of amino acid residues of the desired polypeptideproduct is known. When the entire sequence of amino acid residues of thedesired polypeptide is not known, the direct synthesis of DNA sequencesis not possible and the method of choice is the synthesis of cDNAsequences. Among the standard procedures for isolating cDNA sequences ofinterest is the formation of plasmid- or phage-carrying cDNA librarieswhich are derived from reverse transcription of mRNA which is abundantin donor cells that have a high level of genetic expression. When usedin combination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have been denatured into asingle-stranded form (Jay, et al., Nucl. Acid Res., 11:2325, 1983).

A cDNA expression library, such as lambda gt11, can be screenedindirectly for GDF-11 peptides having at least one epitope, usingantibodies specific for GDF-11. Such antibodies can be eitherpolyclonally or monoclonally derived and used to detect expressionproduct indicative of the presence of GDF-11 cDNA.

DNA sequences encoding GDF-11 can be expressed in vitro by DNA transferinto a suitable host cell. “Host cells” are cells in which a vector canbe propagated and its DNA expressed; The term also includes any progenyof the subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term“host cell” is used. Methods of stable transfer, meaning that theforeign DNA is continuously maintained in the host, are known in theart.

In the present invention, the GDF-11 polynucleotide sequences may beinserted into a recombinant expression vector. The term “recombinantexpression vector” refers to a plasmid, virus or other vehicle known inthe art that has been manipulated by insertion or incorporation of theGDF-11 genetic sequences. Such expression vectors contain a promotersequence which facilitates the efficient transcription of the insertedgenetic sequence of the host. The expression vector typically containsan origin of replication, a promoter, as well as specific genes whichallow phenotypic selection of the transformed cells. Vectors suitablefor use in the present invention include, but are not limited to theT7-based expression vector for expression in bacteria (Rosenberg, etal., Gene, 56:125, 19117), the pMSXND expression vector for expressionin mammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter (e.g., T7, metallothionein 1, orpolyhedrin promoters).

Polynucleotide sequences encoding GDF-11 can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art. Such vectorsare used to incorporate DNA sequences of the invention. Preferably, themature C-terminal region of GDF-11 is expressed from a cDNA clonecontaining the entire coding sequence of GDF-11. Alternatively, theC-terminal portion of GDF-11 can be expressed as a fusion protein withthe pro-region of another member of the TGF-β family or co-expressedwith another pro-region (see for example, Hammonds, et al., Molec.Endocrinol., 5:149, 1991; Gray, A., and Mason, A., Science, 247:1328,1990).

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂ or RbCl canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the GDF-11 of the invention,and a second foreign DNA molecule encoding a selectable phenotype, suchas the herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein. (see for example, Eukaryotic Viral Vectors, ColdSpring Harbor Laboratory, Gluzman ed., 1982).

Isolation and purification of microbial expressed polypeptide, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

GDF-11 Antibodies and Methods of Use

The invention includes antibodies immunoreactive with GDF-11 polypeptideor functional fragments thereof. Antibody which consists essentially ofpooled monoclonal antibodies with different epitopic specificities, aswell as distinct monoclonal antibody preparations are provided.Monoclonal antibodies are made from antigen containing fragments of theprotein by methods well known to those skilled in the art (Kohler, etal., Nature, 256:495, 1975). The term antibody as used in this inventionis meant to include intact molecules as well as fragments thereof, suchas Fab and F(ab′)₂, Fv and SCA fragments which are capable of binding anepitopic determinant on GDF-11.

(1) An Fab fragment consists of a monovalent antigen-binding fragment ofan antibody molecule, and can be produced by digestion of a wholeantibody molecule with the enzyme papain, to yield a fragment consistingof an intact light chain and a portion of a heavy chain.

(2) An Fab′ fragment of an antibody molecule can be obtained by treatinga whole antibody molecule with pepsin, followed by reduction, to yield amolecule consisting of an intact light chain and a portion of a heavychain. Two Fab′ fragments are obtained per antibody molecule treated inthis manner.

(3) An (Fab′)₂ fragment of an antibody can be obtained by treating awhole antibody molecule with the enzyme pepsin, without subsequentreduction. A (Fab′)₂ fragment is a dimer of two Fab′ fragments, heldtogether by two disulfide bonds.

(4) An Fv fragment is defined as a genetically engineered fragmentcontaining the variable region of a light chain and the variable regionof a heavy chain expressed as two chains.

(5) A single chain antibody (“SCA”) is a genetically engineered singlechain molecule containing the variable region of a light chain and thevariable region of a heavy chain, linked by a suitable, flexiblepolypeptide linker.

As used in this invention, the term “epitope” refers to an antigenicdeterminant on an antigen, such as a GDF-11 polypeptide, to which theparatope of an antibody, such as an GDF-11-specific antibody, binds.Antigenic determinants usually consist of chemically active surfacegroupings of molecules, such as amino acids or sugar side chains, andcan have specific three-dimensional structural characteristics, as wellas specific charge characteristics.

As is mentioned above, antigens that can be used in producingGDF-11-specific antibodies include GDF-11 polypeptides or GDF-11polypeptide fragments. The polypeptide or peptide used to immunize ananimal can be obtained by standard recombinant, chemical synthetic, orpurification methods. As is well known in the art, in order to increaseimmunogenicity, an antigen can be conjugated to a carrier protein.Commonly used carriers include keyhole limpet hemocyanin (KLH),thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. Thecoupled peptide is then used to immunize the animal (e.g., a mouse, arat, or a rabbit). In addition to such carriers, well known adjuvantscan be administered with the antigen to facilitate induction of a strongimmune response.

The term “cell-proliferative disorder” denotes malignant as well asnon-malignant cell populations which often appear to differ from thesurrounding tissue both morphologically and genotypically. Malignantcells (i.e., cancer) develop as a result of a multistep process. TheGDF-11 polynucleotide that is an antisense molecule or that encodes adominant negative GDF-11 is useful in treating malignancies of thevarious organ systems, particularly, for example, cells in muscle, bone,kidney or adipose tissue. Essentially, any disorder which isetiologically linked to altered expression of GDF-11 could be consideredsusceptible to treatment with a GDF-11 agent (e.g., a suppressing orenhancing agent). One such disorder is a malignant cell proliferativedisorder, for example.

The invention provides a method for detecting a cell proliferativedisorder of muscle, bone, kidney, uterine or neural tissue, for example,which comprises contacting an anti-GDF-11 antibody with a cell suspectedof having a GDF-11 associated disorder and detecting binding to theantibody. The antibody reactive with GDF-11 is labeled with a compoundwhich allows detection of binding to GDF-11. For purposes of theinvention, an antibody specific for GDF-11 polypeptide may be used todetect the level of GDF-11 in biological fluids and tissues. Anyspecimen containing a detectable amount of antigen can be used. Apreferred sample of this invention is muscle, bone, kidney, uterus,spleen, thymus, or neural tissue. The level of GDF-11 in the suspectcell can be compared with the level in a normal cell to determinewhether the subject has a GDF-11-associated cell proliferative disorder.Such methods of detection are also useful using nucleic acidhybridization to detect the level of GDF-11 mRNA in a sample or todetect an altered GDF-11 gene. Preferably the subject is human.

The antibodies of the invention can be used in any subject in which itis desirable to administer in vitro or in vivo immunodiagnosis orimmunotherapy. The antibodies of the invention are suited for use, forexample, in immunoassays in which they can be utilized in liquid phaseor bound to a solid phase carrier. In addition, the antibodies in theseimmunoassays can be detectably labeled in various ways. Examples oftypes of immunoassays which can utilize antibodies of the invention arecompetitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA) and the sandwich (immunometric) assay. Detection of the antigensusing the antibodies of the invention can be done utilizing immunoassayswhich are run in either the forward, reverse, or simultaneous modes,including immunohistochemical assays on physiological samples. Those ofskill in the art will know, or can readily discern, other immunoassayformats without undue experimentation.

The antibodies of the invention can be bound to many different carriersand used to detect the presence of an antigen comprising the polypeptideof the invention. Examples of well-known carriers include glass,polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding antibodies, or will be able toascertain such, using routine experimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds,phosphorescent compounds, and bioluminescent compounds. Those ofordinary skill in the art will know of other suitable labels for bindingto the antibody, or will be able to ascertain such, using routineexperimentation.

Another technique which may also result in greater sensitivity consistsof coupling the antibodies to low molecular weight haptens. Thesehaptens can then be specifically detected by means of a second reaction.For example, it is common to use such haptens as biotin, which reactswith avidin, or dinitrophenyl, puridoxal, and fluorescein, which canreact with specific antihapten antibodies.

In using the monoclonal antibodies of the invention for the in vivodetection of antigen, the detectably labeled antibody is given a dosewhich is diagnostically effective. The term “diagnostically effective”means that the amount of detectably labeled monoclonal antibody isadministered in sufficient quantity to enable detection of the sitehaving the antigen comprising a polypeptide of the invention for whichthe monoclonal antibodies are specific.

The concentration of detectably labeled monoclonal antibody which isadministered should be sufficient such that the binding to those cellshaving the polypeptide is detectable compared to the background.Further, it is desirable that the detectably labeled monoclonal antibodybe rapidly cleared from the circulatory system in order to give the besttarget-to-background signal ratio.

As a rule, the dosage of detectably labeled monoclonal antibody for invivo diagnosis will vary depending on such factors as age, sex, andextent of disease of the individual. Such dosages may vary, for example,depending on whether multiple injections are given, antigenic burden,and other factors known to those of skill in the art.

For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that deleterious radiation withrespect to the host is minimized. Ideally, a radioisotope used for invivo imaging will lack a particle emission, but produce a large numberof photons in the 140-250 keV range, which may readily be detected byconventional gamma cameras.

For in vivo diagnosis radioisotopes may be bound to immunoglobulineither directly or indirectly by using an intermediate functional group.intermediate functional groups which often are used to bindradioisotopes which exist as metallic ions to immunoglobulins are thebifunctional chelating agents such as diethylenetriaminepentacetic acid(DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.Typical examples of metallic ions which can be bound to the monoclonalantibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, and²⁰¹T1.

The monoclonal antibodies of the invention can also be labeled with aparamagnetic isotope for purposes of in vivo diagnosis, as in magneticresonance imaging (MRI) or electron spin resonance (ESR). In general,any conventional method for visualizing diagnostic imaging can beutilized. Usually gamma and positron emitting radioisotopes are used forcamera imaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr,and ⁵⁶Fe.

The monoclonal antibodies of the invention can be used in vitro and invivo to monitor the course of amelioration of a GDF-11-associateddisease in a subject. Thus, for example, by measuring the increase ordecrease in the number of cells expressing antigen comprising apolypeptide of the invention or changes in the concentration of suchantigen present in various body fluids, it would be possible todetermine whether a particular therapeutic regimen aimed at amelioratingthe GDF-11-associated disease is effective. The term “ameliorate”denotes a lessening of the detrimental effect of the GDF-11-associateddisease in the subject receiving therapy.

Additional Methods of Treatment and Diagnosis

The present invention identifies a nucleotide sequence that can beexpressed in an altered manner as compared to expression in a normalcell, therefore it is possible to design appropriate therapeutic ordiagnostic techniques directed to this sequence. Treatment includesadministration of a reagent which modulates activity. The term“modulate” envisions the suppression or expression of GDF-11 when it isover-expressed, or augmentation of GDF-11 expression when it isunderexpressed. When a muscle-associated disorder is associated withGDF-11 overexpression, such suppressive reagents as antisense GDF-11polynucleotide sequence, dominant negative sequences or GDF-11 bindingantibody can be introduced into a cell. In addition, an anti-idiotypeantibody which binds to a monoclonal antibody which binds GDF-11 of theinvention, or an epitope thereof, may also be used in the therapeuticmethod of the invention. Alternatively, when a cell proliferativedisorder is associated with underexpression or expression of a mutantGDF-11 polypeptide, a sense polynucleotide sequence (the DNA codingstrand) or GDF-11 polypeptide can be introduced into the cell. Suchmuscle-associated disorders include cancer, muscular dystrophy, spinalcord injury, traumatic injury, congestive obstructive pulmonary disease(COPD), AIDS or cachexia. Neurodegenerative, musculoskeletal, and kidneydisorders are also envisioned as treated by the method of the invention.In addition, the method of the invention can be used in the treatment ofobesity or of disorders related to abnormal proliferation of adipocytes.One of skill in the art can determine whether or not a particulartherapeutic course of treatment is successful by several methodsdescribed herein (e.g., muscle fiber analysis or biopsy; determinationof fat content). The present examples demonstrate that the methods ofthe invention are useful for decreasing fat content, and therefore wouldbe useful in the treatment of obesity and related disorders (e.g.,diabetes).

Thus, where a cell-proliferative disorder is associated with theexpression of GDF-11, nucleic acid sequences that interfere with GDF-11expression at the translational level can be used. This approachutilizes, for example, antisense nucleic acid and ribozymes to blocktranslation of a specific GDF-11 mRNA, either by masking that mRNA withan antisense nucleic acid or by cleaving it with a ribozyme. Suchdisorders include neurodegenerative diseases, for example. In addition,dominant-negative GDF-11 mutants would be useful to actively interferewith function of “normal” GDF-11.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (Weintraub, ScientificAmerican, 262:40, 1990). In the cell, the antisense nucleic acidshybridize to the corresponding mRNA, forming a double-stranded molecule.The antisense nucleic acids interfere with the translation of the mRNA,since the cell will not translate a mRNA that is double-stranded.

Antisense oligomers of about 15 nucleotides are preferred, since theyare easily synthesized and are less likely to cause problems than largermolecules when introduced into the target GDF-11-producing cell. The useof antisense methods to inhibit the in vitro translation of genes iswell known in the art (Marcus-Sakura, Anal. Biochem., 172:289, 1988).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

In another embodiment of the present invention, a nucleotide sequenceencoding a GDF-11 dominant negative protein is provided. For example, agenetic construct that contain such a dominant negative encoding genemay be operably linked to a promoter, such as a tissue-specificpromoter. For example, a skeletal muscle specific promoter (e.g., humanskeletal muscle α-actin promoter) or developmentally specific promoter(e.g., MyHC 3, which is restricted in skeletal muscle to the embryonicperiod of development, or an inducible promoter (e.g., the orphannuclear receptor TIS1).

Such constructs are useful in methods of modulating a subject's skeletalmass. For example, a method include transforming an organism, tissue,organ or cell with a genetic construct encoding a dominant negativeGDF-11 protein and suitable promoter in operable linkage and expressingthe dominant negative encoding GDF-11 gene, thereby modulating musclemass, bone content and/or kidney growth by interfering with wild-typeGDF-11 activity.

GDF-11 most likely forms dimers, homodimers or heterodimers and may evenform heterodimers with other GDF family members, such as GDF-11 (seeExample 4). Hence, while not wanting to be bound by a particular theory,the dominant negative effect described herein may involve the formationof non-functional homodimers or heterodimers of dominant negative andwild-type GDF-11 monomers. More specifically, it is possible that anynon-functional homodimer or any heterodimer formed by the dimerizationof wild-type and dominant negative GDF-11 monomers produces a dominanteffect by: 1) being synthesized but not processed or secreted; 2)inhibiting the secretion of wild type GDF-11; 3) preventing normalproteolytic cleavage of the preprotein thereby producing anon-functional GDF-11 molecule; 4) altering the affinity of thenon-functional dimer (e.g., homodimeric or heterodimeric GDF-11) to areceptor or generating an antagonistic form of GDF-11 that binds areceptor without activating it; or 5) inhibiting the intracellularprocessing or secretion of GDF-11 related or TGF-β family proteins.

Non-functional GDF-11 can function to inhibit the growth regulatingactions of GDF-11 on muscle cells that include a dominant negativeGDF-11 gene. Deletion or missense dominant negative forms of GDF-11 thatretain the ability to form dimers with wild-type GDF-11 protein but donot function as wild-type GDF-11 proteins may be used to inhibit thebiological activity of endogenous wild-type GDF-11. For example, in oneembodiment, the proteolytic processing site of GDF-11 may be altered(e.g., deleted) resulting in a GDF-11 molecule able to under subsequentdimerization with endogenous wild-type GDF-11 but unable to undergofurther processing into a mature GDF-11 form. Alternatively, anon-functional GDF-11 can function as a monomeric species to inhibit thegrowth regulating actions of GDF-11 on muscle cells, bone cells andkidney cells at any point in a tissue's or organism's development.

Any genetic recombinant method in the art may be used, for example,recombinant viruses may be engineered to express a dominant negativefrom of GDF-11 which may be sued to inhibit the activity of wild-typeGDF-11. Such viruses may be used therapeutically for treatment ofdiseases resulting from aberrant over-expression or activity of GDF-11protein, such as in denervation hypertrophy or as a means of controllingGDF-11 expression when treating disease conditions involving muscle,such as in musculodegenerative diseases or in tissue repair due totrauma or in modulating GDF-11 expression in animal husbandry (e.g.,transgenic animals for agricultural purposes).

In addition, the expression of GDF-11 may be used, for example to helpin kidney development. The method includes administering atherapeutically effective amount of a GDF-11 agent to the subject,thereby promoting kidney cell growth and differentiation in kidneytissue. The agent may be an antagonist or agonist of GDF-11 activity.For example, the agent may include a GDF-11 antisense molecule or adominant negative polypeptide.

The invention provides a method for treating a muscle, kidney (chronicor acute) or adipose tissue disorder in a subject. The method includesadministering a therapeutically effective amount of a GDF-11 agent tothe subject, thereby inhibiting abnormal growth of muscle or adiposetissue or stimulating growth in kidney tissue. The GDF-11 agent mayinclude a GDF-11 antisense molecule or a dominant negative polypeptide,for example. A “therapeutically effective amount” of a GDF-11 agent isthat amount that ameliorates symptoms of the disorder or inhibits GDF-11induced growth of muscle, for example, as compared with a normalsubject.

The present invention also provides gene therapy for the treatment ofcell proliferative or immunologic disorders which are mediated by GDF-11protein. Such therapy would achieve its therapeutic effect byintroduction of the GDF-11 antisense polynucleotide or dominant negativeencoding polynucleotide sequences into cells having the proliferativedisorder. Delivery of antisense GDF-11 polynucleotide can be achievedusing a recombinant expression vector such as a chimeric virus or acolloidal dispersion system. Especially preferred for therapeuticdelivery of antisense or dominant negative sequences is the use oftargeted liposomes. In contrast, when it is desirable to enhance GDF-11production, a “sense” GDF-11 polynucleotide is introduced into theappropriate cell(s).

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. By inserting a GDF-11 sequence of interestinto the viral vector, along with another gene which encodes the ligandfor a receptor on a specific target cell, for example, the vector is nowtarget specific. Retroviral vectors can be made target specific byattaching, for example, a sugar, a glycolipid, or a protein. Preferredtargeting is accomplished by using an antibody to target the retroviralvector. Those of skill in the art will know of, or can readily ascertainwithout undue experimentation, specific polynucleotide sequences whichcan be inserted into the retroviral genome or attached to a viralenvelope to allow target specific delivery of the retroviral vectorcontaining the GDF-11 antisense polynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsulation. Helper cell lines which havedeletions of the packaging signal include, but are not limited to ψ2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced.

Alternatively, NIH 3T3 or other tissue culture cells can be directlytransfected with plasmids encoding the retroviral structural genes gag,pol and env, by conventional calcium phosphate transfection. These cellsare then transfected with the vector plasmid containing the genes ofinterest. The resulting cells release the retroviral vector into theculture medium.

Another targeted delivery system for GDF-11 antisense polynucleotides isa colloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. It has beenshown that large unilamellar vesicles (LUV), which range in size from0.2-4.0 μm can encapsulate a substantial percentage of an aqueous buffercontaining large macromolecules. RNA, DNA and intact virions can beencapsulated within the aqueous interior and be delivered to cells in abiologically active form (Fraley, et al., Trends Biochem. Sci., 6:77,19111). In addition to mammalian cells, liposomes have been used fordelivery of polynucleotides in plant, yeast and bacterial cells. inorder for a liposome to be an efficient gene transfer vehicle, thefollowing characteristics should be present: (1) encapsulation of thegenes of interest at high efficiency while not compromising theirbiological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Manning, et al., BioTechniques, 6:682, 1988).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

Due to the expression of GDF-11 in muscle and adipose tissue, there area variety of applications using the polypeptide, polynucleotide, andantibodies of the invention, related to these tissues. Such applicationsinclude treatment of cell proliferative disorders involving these andother tissues, such as neural tissue. In addition, GDF-11 may be usefulin various gene therapy procedures. In embodiments where GDF-11polypeptide is administered to a subject, the dosage range is about 0.1μg/kg to 100 mg/kg; more preferably from about 1 μg/kg to 75 mg/kg andmost preferably from about 10 mg/kg to 50 mg/kg.

Chromosomal Location of GDF-11

The data in Example 6 shows that the human GDF-11 gene is located onchromosome 2. By comparing the chromosomal location of GDF-11 with themap positions of various human disorders, it should be possible todetermine whether mutations in the GDF-11 gene are involved in theetiology of human diseases. For example, an autosomal recessive form ofjuvenile amyotrophic lateral sclerosis has been shown to map tochromosome 2 (Hentati, et al., Neurology, 42 (Suppl.3):201, 1992). Moreprecise mapping of GDF-11 and analysis of DNA from these patients mayindicate that GDF-11 is, in fact, the gene affected in this disease. Inaddition, GDF-11 is useful for distinguishing chromosome 2 from otherchromosomes.

Transgenic Animals and Methods of Making the Same

Various methods to make the transgenic animals of the subject inventioncan be employed. Generally speaking, three such methods may be employed.In one such method, an embryo at the pronuclear stage (a “one cellembryo”) is harvested from a female and the transgene is microinjectedinto the embryo, in which case the transgene will be chromosomallyintegrated into both the germ cells and somatic cells of the resultingmature animal. In another such method, embryonic stem cells are isolatedand the transgene incorporated therein by electroporation, plasmidtransfection or microinjection, followed by reintroduction of the stemcells into the embryo where they colonize and contribute to the germline. Methods for microinjection of mammalian species is described inU.S. Pat. No. 4,873,191. In yet another such method, embryonic cells areinfected with a retrovirus containing the transgene whereby the germcells of the embryo have the transgene chromosomally integrated therein.When the animals to be made transgenic are avian, because avianfertilized ova generally go through cell division for the first twentyhours in the oviduct, microinjection into the pronucleus of thefertilized egg is problematic due to the inaccessibility of thepronucleus. Therefore, of the methods to make transgenic animalsdescribed generally above, retrovirus infection is preferred for avianspecies, for example as described in U.S. Pat. No. 5,162,215. Ifmicroinjection is to be used with avian species, however, a recentlypublished procedure by Love et al. (BioTechnology, 12, Jan. 1994) can beutilized whereby the embryo is obtained from a sacrificed henapproximately two and one-half hours after the laying of the previouslaid egg, the transgene is microinjected into the cytoplasm of thegerminal disc and the embryo is cultured in a host shell until maturity.When the animals to be made transgenic are bovine or porcine,microinjection can be hampered by the opacity of the ova thereby makingthe nuclei difficult to identify by traditional differentialinterference-contrast microscopy. To overcome this problem, the ova canfirst be centrifuged to segregate the pronuclei for bettervisualization.

The “non-human animals” of the invention bovine, porcine, ovine andavian animals (e.g., cow, pig, sheep, chicken). The “transgenicnon-human animals” of the invention are produced by introducing“transgenes” into the germline of the non-human animal. Embryonal targetcells at various developmental stages can be used to introducetransgenes. Different methods are used depending on the stage ofdevelopment of the embryonal target cell. The zygote is the best targetfor microinjection. The use of zygotes as a target for gene transfer hasa major advantage in that in most cases the injected DNA will beincorporated into the host gene before the first cleavage (Brinster etal., Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985). As a consequence,all cells of the transgenic non-human animal will carry the incorporatedtransgene. This will in general also be reflected in the efficienttransmission of the transgene to offspring of the founder since 50% ofthe germ cells will harbor the transgene.

The term “transgenic” is used to describe an animal which includesexogenous genetic material within all of its cells. A “transgenic”animal can be produced by cross-breeding two chimeric animals whichinclude exogenous genetic material within cells used in reproduction.Twenty-five percent of the resulting offspring will be transgenic i.e.,animals which include the exogenous genetic material within all of theircells in both alleles. 50% of the resulting animals will include theexogenous genetic material within one allele and 25% will include noexogenous genetic material.

In the microinjection method useful in the practice of the subjectinvention, the transgene is digested and purified free from any vectorDNA e.g. by gel electrophoresis. It is preferred that the transgeneinclude an operatively associated promoter which interacts with cellularproteins involved in transcription, ultimately resulting in constitutiveexpression. Promoters useful in this regard include those fromcytomegalovirus (CMV), Moloney leukemia virus (MLV), and herpes virus,as well as those from the genes encoding metallothionein, skeletalactin, P-enolpyruvate carboxylase (PEPCK), phosphoglycerate (PGK), DHFR,and thymidine kinase. Promoters for viral long terminal repeats (LTRs)such as Rous Sarcoma Virus can also be employed. When the animals to bemade transgenic are avian, preferred promoters include those for thechicken β-globin gene, chicken lysozyme gene, and avian leukosis virus.Constructs useful in plasmid transfection of embryonic stem cells willemploy additional regulatory elements well known in the art such asenhancer elements to stimulate transcription, splice acceptors,termination and polyadenylation signals, and ribosome binding sites topermit translation.

Retroviral infection can also be used to introduce transgene into anon-human animal, as described above. The developing non-human embryocan be cultured in vitro to the blastocyst stage. During this time, theblastomeres can be targets for retro viral infection (Jaenich, R., Proc.Natl. Acad. Sci USA 73:1260-1264, 1976). Efficient infection of theblastomeres is obtained by enzymatic treatment to remove the zonapellucida (Hogan, et al. (1986) in Manipulating the Mouse Embryo, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The viralvector system used to introduce the transgene is typically areplication-defective retrovirus carrying the transgene (Jahner, et al.,Proc. Natl. Acad. Sci. USA 82:6927-6931, 1985; Van der Putten, et al.,Proc. Natl. Acad. Sci USA 82:6148-6152, 1985). Transfection is easilyand efficiently obtained by culturing the blastomeres on a monolayer ofvirus-producing cells (Van der Putten, supra; Stewart et al., EMBO J.6:383-388, 1987). Alternatively, infection can be performed at a laterstage. Virus or virus-producing cells can be injected into theblastocoele (D. Jahner et al., Nature 298:623-628, 1982). Most of thefounders will be mosaic for the transgene since incorporation occursonly in a subset of the cells which formed the transgenic nonhumananimal. Further, the founder may contain various retro viral insertionsof the transgene at different positions in the genome which generallywill segregate in the offspring. In addition, it is also possible tointroduce transgenes into the germ line, albeit with low efficiency, byintrauterine retroviral infection of the midgestation embryo (D. Jahneret al., supra).

A third type of target cell for transgene introduction is the embryonalstem cell (ES). ES cells are obtained from pre-implantation embryoscultured in vitro and fused with embryos (Evans et al. Nature292:154-156, 1981; Bradley et al., Nature 309: 255-258, 1984; Gossler,et al., Proc. Natl. Acad. Sci USA 83:9065-9069, 1986; and Robertson etal., Nature 322:445-448, 1986). Transgenes can be efficiently introducedinto the ES cells by DNA transfection or by retro virus-mediatedtransduction. Such transformed ES cells can thereafter be combined withblastocysts from a nonhuman animal. The ES cells thereafter colonize theembryo and contribute to the germ line of the resulting chimeric animal.(For review see Jaenisch, R., Science 240:1468-1474, 1988).

“Transformed” means a cell into which (or into an ancestor of which) hasbeen introduced, by means of recombinant nucleic acid techniques, aheterologous nucleic acid molecule. “Heterologous” refers to a nucleicacid sequence that either originates from another species or is modifiedfrom either its original form or the form primarily expressed in thecell.

“Transgene” means any piece of DNA which is inserted by artifice into acell, and becomes part of the genome of the organism (i.e., eitherstably integrated or as a stable extrachromosomal element) whichdevelops from that cell. Such a transgene may include a gene which ispartly or entirely heterologous (i.e., foreign) to the transgenicorganism, or may represent a gene homologous to an endogenous gene ofthe organism. Included within this definition is a transgene created bythe providing of an RNA sequence which is transcribed into DNA and thenincorporated into the genome. The transgenes of the invention includeDNA sequences which encode GDF-11, and include GDF-sense and antisensepolynucleotides and dominant negative encoding polynucleotides, whichmay be expressed in a transgenic non-human animal. The term “transgenic”as used herein additionally includes any organism whose genome has beenaltered by in vitro manipulation of the early embryo or fertilized eggor by any transgenic technology to induce a specific gene knockout. Theterm “gene knockout” as used herein, refers to the targeted disruptionof a gene in vivo with complete loss of function that has been achievedby any transgenic technology familiar to those in the art. In oneembodiment, transgenic animals having gene knockouts are those in whichthe target gene has been rendered nonfunctional by an insertion targetedto the gene to be rendered non-functional by homologous recombination.As used herein, the term “transgenic” includes any transgenic technologyfamiliar to those in the art which can produce an organism carrying anintroduced transgene or one in which an endogenous gene has beenrendered non-functional or “knocked out.” An example of a transgene usedto “knockout” GDF-11 function in the present Examples is described inExample 6 and FIG. 9. Thus, in another embodiment, the inventionprovides a transgene wherein the entire mature C-terminal region ofGDF-11 is deleted.

The transgene to be used in the practice of the subject invention is aDNA sequence comprising a modified GDF-11 coding sequence. In apreferred embodiment, the GDF-11 gene is disrupted by homologoustargeting in embryonic stem cells. For example, the entire matureC-terminal region of the GDF-11 gene may be deleted as described in theexamples below. Optionally, the GDF-11 disruption or deletion may beaccompanied by insertion of or replacement with other DNA sequences,such as a non-functional GDF-11 sequence. In other embodiments, thetransgene comprises DNA antisense to the coding sequence for GDF-11. Inanother embodiment, the transgene comprises DNA encoding an antibody orreceptor peptide sequence which is able to bind to GDF-11. The DNA andpeptide sequences of GDF-11 are known in the art, the sequences,localization and activity disclosed in WO95/08543 and U.S. Ser. No.08/706,958, filed on Sep. 3, 1996, incorporated by reference in itsentirety. The disclosure of both of these applications are herebyincorporated herein by reference. Where appropriate, DNA sequences thatencode proteins having GDF-11 activity but differ in nucleic acidsequence due to the degeneracy of the genetic code may also be usedherein, as may truncated forms, allelic variants and interspecieshomologues.

Therefore the invention also includes animals having heterozygousmutations in GDF-11. A heterozygote would likely have an intermediateincrease in muscle mass as compared to the homozygote.

After an embryo has been microinjected, colonized with transfectedembryonic stem cells or infected with a retrovirus containing thetransgene (except for practice of the subject invention in avian specieswhich is addressed elsewhere herein) the embryo is implanted into theoviduct of a pseudopregnant female. The consequent progeny are testedfor incorporation of the transgene by Southern blot analysis of bloodsamples using transgene specific probes. PCR is particularly useful inthis regard. Positive progeny (G0) are crossbred to produce offspring(G1) which are analyzed for transgene expression by northern blotanalysis of tissue samples. To be able to distinguish expression oflike-species transgenes from expression of the animals endogenous GDF-11gene(s), a marker gene fragment can be included in the construct in the3′ untranslated region of the transgene and the northern probe designedto probe for the marker gene fragment. The serum levels of GDF-11 canalso be measured in the transgenic animal to establish appropriateexpression. Expression of the GDF-11 transgenes, thereby decreasing theGDF-11 in the tissue and serum levels of the transgenic animals andconsequently increasing the muscle tissue content results in thefoodstuffs from these animals (i.e., eggs, beef, pork, poultry meat,milk, etc.) having markedly increased muscle content, and preferablywithout increased, and more preferably, reduced levels of fat andcholesterol. By practice of the subject invention, a statisticallysignificant increase in muscle content, preferably at least a 2%increase in muscle content (e.g., in chickens), more preferably a 25%increase in muscle content as a percentage of body weight, morepreferably greater than 40% increase in muscle content in thesefoodstuffs can be obtained.

In addition decrease in GDF-11 in the tissue and serum levels of thetransgenic animals can be used to increase the bone content of animalsuse as foodstuffs, for example the transgenic animals having reducedGDF-11 can be provided to have an additional number of ribs.

Additional Methods of Use

Thus, the present invention includes methods for increasing muscle massand/or rib content in domesticated animals, characterized byinactivation or deletion of the gene encoding growth and differentiationfactor-11 (GDF-11). The domesticated animal is preferably selected fromthe group consisting of ovine, bovine, porcine, piscine and avian. Theanimal may be treated with an isolated polynucleotide sequence encodingGDF-11 which polynucleotide sequence is also from a domesticated animalselected from the group consisting of ovine, bovine, porcine, piscineand avian. The present invention includes methods for increasing themuscle mass or rib content in domesticated animals characterized byadministering to a domesticated animal monoclonal antibodies directed tothe GDF-11 polypeptide. The antibody may be an anti-GDF-11, and may beeither a monoclonal antibody or a polyclonal antibody.

The invention includes methods comprising using an anti-GDF-11monoclonal antibody, antisense, or dominant negative mutants as atherapeutic agent to inhibit the growth regulating actions of GDF-11 onmuscle cells. Muscle cells are defined to include fetal or adult musclecells, as well as progenitor cells which are capable of differentiationinto muscle. The monoclonal antibody may be a humanized (e.g., eitherfully or a chimeric) monoclonal antibody, of any species origin, such asmurine, ovine, bovine, porcine or avian. Methods of producing antibodymolecules with various combinations of “humanized” antibodies are wellknown in the art and include combining murine variable regions withhuman constant regions (Cabily, et al., Proc. Natl. Acad. Sci. USA,81:3273, 1984), or by grafting the murine-antibody complementarydetermining regions (CDRs) onto the human framework (Richmann et al.,Nature 332:323, 1988). Other general references which teach methods forcreating humanized antibodies include Morrison et al., Science,229:1202, 1985; Jones et al., Nature, 321:522, 1986; Monroe et al.,Nature 312:779, 1985; Oi et al., BioTechniques, 4:214, 1986; EuropeanPatent Application No. 302,620; and U.S. Pat. No. 5,024,834. therefore,by humanizing the monoclonal antibodies of the invention for in vivouse, an immune response to the antibodies would be greatly reduced.

The invention includes methods comprising using an anti-GDF-11monoclonal antibody, antisense, or dominant negative mutants as atherapeutic agent to inhibit the growth regulating actions of GDF-11 onbone cells. Bone cells are defined to include fetal or adult bone cells,as well as progenitor cells which are capable of differentiation intobone. The monoclonal antibody may be a humanized (e.g., either fully ora chimeric) monoclonal antibody, of any species origin, such as murine,ovine, bovine, porcine or avian. Methods of producing antibody moleculeswith various combinations of “humanized” antibodies are well known inthe art and include combining murine variable regions with humanconstant regions (Cabily et al., Proc. Natl. Acad. Sci. USA, 81:3273,1984), or by grafting the murine-antibody complementary determiningregions (CDRs) onto the human framework (Richmann et al., Nature332:323, 1988). Other general references which teach methods forcreating humanized antibodies include Morrison et al., Science,229:1202, 1985; Jones et al., Nature, 321:522, 1986; Monroe et al.,Nature 312:779, 1985; Oi et al., BioTechniques, 4:214, 1986; EuropeanPatent Application No. 302,620; and U.S. Pat. No. 5,024,834. Therefore,by humanizing the monoclonal antibodies of the invention for in vivouse, an immune response to the antibodies would be greatly reduced.

The monoclonal antibody, GDF-11 polypeptide, or GDF-11 polynucleotide(all “GDF-11 agents”) may have the effect of increasing the developmentof skeletal muscles or skeletal bones. In preferred embodiments of theclaimed methods, the GDF-11 monoclonal antibody, polypeptide, orpolynucleotide is administered to a patient suffering from a disorderselected from the group consisting of muscle wasting disease,neuromuscular disorder, muscle atrophy or aging. In another preferredembodiment the invention provides a method for treating bonedegenerative disorders, such as osteoporosis by administering to apatient suffering from a disorder antibodies, polypeptides orpolynucleotides effecting GDF-11 activity. The GDF-11 agent may also beadministered to a patient suffering from a disorder selected from thegroup consisting of muscular dystrophy, spinal cord injury, traumaticinjury, congestive obstructive pulmonary disease (COPD), AIDS orcachexia.

In a preferred embodiment, the GDF-11 agent is administered to a patientwith muscle or bone wasting disease or disorder by intravenous,intramuscular or subcutaneous injection; preferably, a monoclonalantibody is administered within a dose range between about 0.1 mg/kg toabout 100 mg/kg; more preferably between about 1 μg/kg to 75 mg/kg; mostpreferably from about 10 mg/kg to 50 mg/kg. The antibody may beadministered, for example, by bolus injunction or by slow infusion. Slowinfusion over a period of 30 minutes to 2 hours is preferred. The GDF-11agent may be formulated in a formulation suitable for administration toa patient. Such formulations are known in the art.

The dosage regimen will be determined by the attending physicianconsidering various factors which modify the action of the GDF-11protein, e.g. amount of tissue desired to be formed, the site of tissuedamage, the condition of the damaged tissue, the size of a wound, typeof damaged tissue, the patient's age, sex, and diet, the severity of anyinfection, time of administration and other clinical factors. The dosagemay vary with the type of matrix used in the reconstitution and thetypes of agent, such as anti-GDF-11 antibodies, to be used in thecomposition. Generally, systemic or injectable administration, such asintravenous (IV), intramuscular (IM) or subcutaneous (Sub-Q) injection.Administration will generally be initiated at a dose which is minimallyeffective, and the dose will be increased over a preselected time courseuntil a positive effect is observed. Subsequently, incremental increasesin dosage will be made limiting such incremental increases to suchlevels that produce a corresponding increase in effect, while takinginto account any adverse affects that may appear. The addition of otherknown growth factors, such as IGF I (insulin like growth factor I),human, bovine, or chicken growth hormone which may aid in increasingmuscle mass, to the final composition, may also affect the dosage. Inthe embodiment where an anti-GDF-11 antibody is administered, theanti-GDF-11 antibody is generally administered within a dose range ofabout 0.1 μg/kg to about 100 mg/kg.; more preferably between about 10mg/kg to 50 mg/kg.

Progress can be monitored by periodic assessment of tissue growth and/orrepair. The progress can be monitored, for example, x-rays,histomorphometric determinations and tetracycline labeling.

Screening for GDF-11 Modulating Compounds

In another embodiment, the invention provides a method for identifying acompound or molecule that modulates GDF-11 protein activity or geneexpression. The method includes incubating components comprising thecompound, GDF-11 polypeptide or with a recombinant cell expressingGDF-11 polypeptide, under conditions sufficient to allow the componentsto interact and determining the effect of the compound on GDF-11activity or expression. The effect of the compound on GDF-11 activitycan be measured by a number of assays, and may include measurementsbefore and after incubating in the presence of the compound. Compoundsthat affect GDF-11 activity or gene expression include peptides,peptidomimetics, polypeptides, chemical compounds and biologic agents.Assays include northern blot analysis of GDF-11 mRNA (for geneexpression), western blot analysis (for protein level) and muscle fiberanalysis (for protein activity).

The above screening assays may be used for detecting the compounds ormolecules that bind to the GDF-11 receptor or GDF-11 polypeptide, inisolating molecules that bind to the GDF-11 gene, for measuring theamount of GDF-11 in a sample, either polypeptide or RNA (mRNA), foridentifying molecules that may act as agonists or antagonists, and thelike. For example, GDF-11 antagonists are useful for treatment ofmuscular and adipose tissue disorders (e.g., obesity).

Incubating includes conditions which allow contact between the testcompound and GDF-11 polypeptide or with a recombinant cell expressingGDF-11 polypeptide. Contacting includes in solution and in solid phase,or in a cell. The test compound may optionally be a combinatoriallibrary for screening a plurality of compounds. Compounds identified inthe method of the invention can be further evaluated, detected, cloned,sequenced, and the like, either in solution or after binding to a solidsupport, by any method usually applied to the detection of a specificDNA sequence such as PCR, oligomer restriction (Saiki et al.,BioTechnology, 3:1008-1012, 1985), allele-specific oligonucleotide (ASO)probe analysis (Conner et al., Proc. Natl. Acad. Sci. USA, 80:278,1983), oligonucleotide Landegren et al., Science, 241:1077, 1988), andthe like. Molecular techniques for DNA analysis have been reviewed(Landegren et al., Science, 242:229-237, 1988).

All references cited herein are hereby incorporated by reference intheir entirety.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLE 1 Identification and Isolation of a Novel TGF-β Family Member

To identify novel members of the TGF-β superfamily, a murine genomiclibrary was screened at reduced stringency using a murine GDF-8 probe(FIG. 8; nucleotides 865-1234) spanning the region encoding theC-terminal portion of the GDF-8 precursor protein. Hybridization wascarried out as described (Lee, Mol. Endocrinol., 4:1034, 1990) at 65°C., and the final wash was carried out at the same temperature in abuffer containing 0.5 M NaCl. Among the hybridizing phage was one thatcould be distinguished from GDF-8-containing phage on the basis of itsreduced hybridization intensity to the GDF-8 probe. Partial nucleotidesequence analysis of the genomic insert present in this weaklyhybridizing phage showed that this clone contained a sequence highlyrelated to but distinct from murine GDF-8.

A partial nucleotide sequence of the genomic insert present in thisphage is shown in FIG. 1A. The sequence contained an open reading frameextending from nucleotides 198 to 575 that showed significant homologyto the known members of the TGF-β superfamily (see below). Precedingthis sequence was a 3′ splice consensus sequence at precisely the sameposition as in the GDF-8 gene. This new TGF-β family member was giventhe designation GDF-11 (growth/differentiation factor-11).

EXAMPLE 2 Expression of GDF-11

To determine the expression pattern of GDF-11, RNA samples prepared froma variety of tissues were screened by northern blot analysis. RNAisolation and northern analysis were carried out as described previously(Lee, Mol. Endocrinol., 4:1034, 1990) except that the hybridization wascarried out in 5×SSPE, 10% dextran sulfate, 50% formamide, 1% SDS, 200μg/ml salmon DNA, and 0.1% each of bovine serum albumin, ficoll, andpolyvinylpyrrolidone. Five micrograms of twice poly A-selected RNAprepared from each tissue (except for 2 day neonatal brain, for whichonly 3.3 μg RNA were used) were electrophoresed on formaldehyde gels,blotted, and probed with GDF-11. As shown in FIG. 2, the GDF-11 probedetected two RNA species, approximately 4.2 and 3.2 kb in length, inadult thymus, brain, spleen, uterus, and muscle as well as in wholeembryos isolated at day 12.5 or 18.5 and in brain samples taken atvarious stages of development. On longer exposures of these blots, lowerlevels of GDF-11 RNA could also be detected in a number of othertissues.

EXAMPLE 3 Isolation of cDNA Clones Encoding GDF-11

In order to isolate cDNA clones encoding GDF-11, a cDNA library wasprepared in the lambda ZAP II vector (Stratagene) using RNA preparedfrom human adult spleen. From 5 μg of twice poly A-selected RNA preparedfrom human spleen, a cDNA library consisting of 21 million recombinantphage was constructed according to the instructions provided byStratagene. The library was screened without amplification. Libraryscreening and characterization of cDNA inserts were carried out asdescribed previously (Lee, Mol. Endocrinol., 4:1034, 1990). From thislibrary, 23 hybridizing phage were obtained.

The entire nucleotide sequence of the clone extending furthest towardthe 5′ end of the gene was determined. The 1258 base pair sequencecontained a single long open reading frame beginning from the 5′ end ofthe clone and extending to a TAA stop codon. Because the open readingframe and the homology with GDF-8 (see below) extended to the very 5′end of the clone, it seemed likely that this clone was missing thecoding sequence corresponding to the N-terminal portion of the GDF-11precursor protein. In order to obtain the remaining portion of theGDF-11 sequence, several genomic clones were isolated by screening ahuman genomic library with the human GDF-11 cDNA probe. Partial sequenceanalysis of one of these genomic clones showed that this clone containedthe GDF-11 gene. From this clone, the remaining GDF-11 coding sequencewas obtained. FIG. 1B shows the predicted sequence of GDF-11 assembledfrom the genomic and cDNA sequences. Nucleotides 136 to 1393 representthe extent of the sequence obtained from a cDNA clone. Nucleotides 1 to135 were obtained from a genomic clone. The sequence has beenarbitrarily numbered beginning with a Sac II site present in the genomicclone, but the location of the mRNA start site is not known. Thesequence contains a putative initiating methionine at nucleotide 54.Whether the sequence upstream of this methionine codon is all present inthe mRNA is not known. Beginning with this methionine codon, the openreading frame extends for 407 amino acids. The sequence contains onepotential N-linked glycosylation site at asparagine 94. The sequencecontains a predicted RXXR (SEQ ID NO:12) proteolytic cleavage site atamino acids 295 to 298, and cleavage of the precursor at this site wouldgenerate an active C-terminal fragment 109 amino acids in length with apredicted molecular weight of approximately 12,500 kD. In this region,the predicted murine and human GDF-11 amino acid sequences are 100%identical. The high degree of sequence conservation across speciessuggests that GDF-11 plays an important role in vivo.

The C-terminal region following the predicted cleavage site contains allthe hallmarks present in other TGF-β family members. GDF-11 containsmost of the residues that are highly conserved in other family members,including the seven cysteine residues with their characteristic spacing.Like the TGF-β's, the inhibin β's, and GDF-8, GDF-11 also contains twoadditional cysteine residues. In the case of TGF-β2, these additionalcysteine residues are known to form an intramolecular disulfide bond(Daopin, et al., Science, 257:369, 1992; Schlunegger and Grutter,Nature, 358:430, 1992). Atabulation of the amino acid sequencehomologies between GDF-11 and the other TGF-β family members is shown inFIG. 3. Numbers represent percent amino acid identities between eachpair calculated from the first conserved cysteine to the C-terminus.Boxes represent homologies among highly-related members withinparticular subgroups. In this region, GDF-11 is most highly related toGDF-8 (92% sequence identity).

An alignment of GDF-8 (SEQ ID NO:5) and GDF-11 (SEQ ID NO:6) amino acidsequences is shown in FIG. 4A. The two sequences contain potentialN-linked glycosylation signals (NIS) and putative proteolytic processingsites (RSRR; SEQ ID NO:11) at analogous positions. The two sequences arerelated not only in the C-terminal region following the putativecleavage site (90% amino acid sequence identity), but also in thepro-region of the molecules (45% amino acid sequence identity).

EXAMPLE 4 Construction of a Hybrid GDF-8/GDF-11 Gene

In order to express GDF-11 protein, a hybrid gene was constructed inwhich the N-terminal region of GDF-11 was replaced by the analogousregion of GDF-8. Such hybrid constructs have been used to producebiologically-active BMP-4 (Hammonds et al., Mol. Endocrinol., 5:149,1991) and Vg-1 (Thomsen and Melton, Cell, 74:433, 1993). In order toensure that the GDF-11 protein produced from the hybrid construct wouldrepresent authentic GDF-11, the hybrid gene was constructed in such amanner that the fusion of the two gene fragments would occur preciselyat the predicted cleavage sites. In particular, an AvaII restrictionsite is present in both sequences at the location corresponding to thepredicted proteolytic cleavage site. The N-terminal pro-region of GDF-8up to this AvaII site was obtained by partial digestion of the clonewith AvaII and fused to the C-terminal region of GDF-11 beginning atthis AvaII site. The resulting hybrid construct was then inserted intothe pMSXND mammalian expression vector (Lee and Nathans, J. Biol. Chem.,263:3521) and transfected into Chinese hamster ovary cells. As shown inFIG. 5, western blot analysis of conditioned medium from G418-resistantcells using antibodies raised against the C-terminal portion of GDF-8showed that these cells secreted GDF-11 protein into the medium and thatat least some of the hybrid protein was proteolytically processed.Furthermore, these studies demonstrate that the antibodies directedagainst the C-terminal portion of GDF-8 will also react with GDF-11protein.

EXAMPLE 5 Chromosomal Localization of GDF-11

In order to map the chromosomal location of GDF-11, DNA samples fromhuman/rodent somatic cell hybrids (Drwinga, et al., Genomics,16:311-313, 1993; Dubois and Naylor, Genomics, 16:315-319, 1993) wereanalyzed by polymerase chain reaction followed by Southern blotting.Polymerase chain reaction was carried out using primer #101,5′-GAGTCCCGCTGCTGCCGATATCC-3′, (SEQ ID NO:7) and primer #102,5′-TAGAGCATGTTGATTGGGGACAT-3′, (SEQ ID NO:8) for 35 cycles at 94° C. for2 minutes, 58° C. for 1 minutes, and 72° C. for 1 minute. These primerscorrespond to nucleotides 981 to 1003 and the reverse complement ofnucleotides 1182 to 1204, respectively, in the human GDF-11 sequence.PCR products were electrophoresed on agarose gels, blotted, and probedwith oligonucleotide #104, 5′-AAATATCCGCATACCCATTT-3′, (SEQ ID NO:9)which corresponds to a sequence internal to the region flanked by primer#101 and #102. Filters were hybridized in 6×SSC, 1× Denhardt's solution,100 μg/ml yeast transfer RNA, and 0.05% sodium pyrophosphate at 50° C.

As shown in FIG. 6, the human-specific probe detected a band of thepredicted size (approximately 224 base pairs) in the positive controlsample (total human genomic DNA) and in a single DNA sample from thehuman/rodent hybrid panel. This positive signal corresponds to humanchromosome 12. The human chromosome contained in each of the hybrid celllines is identified at the top of each of the first 24 lanes (1-22, X,and Y). In the lanes designated CHO, M, and H, the starting DNA templatewas total genomic DNA from hamster, mouse, and human sources,respectively. In the lane marked B1, no template DNA was used. Numbersat left indicate the mobilities of DNA standards. These data show thatthe human GDF-11 gene is located on chromosome 12.

In order to determine the more precise location of GDF-11 on chromosome12, the GDF-11 gene was localized by florescence in situ hybridization(FISH). These FISH localization studies were carried out by contract toBIOS laboratories (New Haven, Conn.). Purified DNA from a human GDF-11genomic clone was labeled with digoxigenin dUTP by nick translation.Labeled probe was combined with sheared human DNA and hybridized tonormal metaphase chromosomes derived from PHA stimulated peripheralblood lymphocytes in a solution containing 50% formamide, 10% dextransulfate and 2×SSC. Specific hybridization signals were detected byincubating the hybridized slides in fluorescein-conjugated sheepantidigoxigenin antibodies. Slides were then counterstained withpropidium iodide and analyzed. As shown in FIG. 7A, this experimentresulted in the specific labeling of the proximal long arm of a group Cchromosome, the size and morphology of which were consistent withchromosome 12. In order to confirm the identity of the specificallylabeled chromosome, a second experiment was conducted in which achromosome 12-specific centromere probe was cohybridized with GDF-11. Asshown in FIG. 7B, this experiment clearly demonstrated that GDF-11 islocated at a position which is 23% of the distance from the centromereto the telomere of the long arm of chromosome 12, an area whichcorresponds to band 12q13 (FIG. 7C). A total of 85 metaphase cells wereanalyzed and 80 exhibited specific labeling.

EXAMPLE 6 GDF-11 Homology in Mammalian Species

Like most other TGF-β family member, GDF-11 also appears to be highlyconserved across species. By genomic Southern analysis, homologoussequences were detected in all mammalian species examined as well as inchickens and frogs (FIG. 4B). In most species, the GDF-11 probe alsodetected a second, more faintly hybridizing fragment corresponding tothe myostatin gene (McPherron et al., 1997).

EXAMPLE 7 GDF-11 Transgenic Knockout Mice

To determine the biological function of GDF-11, we disrupted the GDF-11gene by homologous targeting in embryonic stem cells. A murine 129 SV/Jgenomic library was prepared in lambda FIXII vector according to theinstructions provided by Stratagene (La Jolla, Calif.). The structure ofthe GDF-11 gene was deduced from restriction mapping and partialsequencing of phage clones isolated from the library. Vectors forpreparing the targeting construct were kindly provided by Philip Sorianoand Kirk Thomas. To ensure that the resulting mice would be null forGDF-11 function, the entire mature C-terminal region was deleted andreplaced by a neo cassette (FIGS. 9A and 9B). R1 ES cells weretransfected with the targeting construct, selected with gancyclovir (2μM) and G418 (250 μg/ml), and analyzed by Southern analysis. Homologoustargeting of the GDF-11 gene was seen in 8/155 gancyclovir/G418 doublyresistant ES cell clones. Following injection of several targeted clonesinto C57BL/6J blastocysts, we obtained chimeras from one ES clone thatproduced heterozygous pups when crossed to both C57BL/6J and 129/SvJfemales. Crosses of C57BL/6J/129/SvJ hybrid F1 heterozygotes produced 49wild-type (34%), 94 heterozygous (66%) and no homozygous mutant adultoffspring. Similarly, there were no adult homozygous null animals seenin the 129/SvJ background (32 wild-type (36%) and 56 heterozygous mutant(64%) animals).

To determine the age at which homozygous mutants were dying, wegenotyped litters of embryos isolated at various gestational ages fromheterozygous females that had been mated to heterozygous males. At allembryonic stages examined, homozygous mutant embryos were present atapproximately the predicted frequency of 25%. Among hybrid newborn mice,the different genotypes were also represented at the expected Mendelianratio of 1:2:1 (34+/+ (28%), 61± (50%), and 28 −/− (23%)). Homozygousmutant mice were born alive and were able to breath and nurse. Allhomozygous mutants died, however, within the first 24 hours after birth.The precise cause of death was unknown, but the lethality may have beenrelated to the fact that the kidneys in homozygous mutants were eitherseverely hypoplastic or completely absent. A summary of the kidneyabnormalities in these mice is shown in FIG. 10.

EXAMPLE 8 Anatomical Differences in Knockout Mice

Homozygous mutant animals were easily recognizable by their severelyshortened or absent tails (FIG. 11A). To further characterize the taildefects in these homozygous mutant animals, we examined their skeletonsto determine the degree of disruption of the caudal vertebrae. Acomparison of wild-type and mutant skeleton preparations of late stageembryos and newborn mice, however, revealed differences not only in thecaudal region of the animals but in many other regions as well. Innearly every case where differences were noted, the abnormalitiesappeared to represent homeotic transformations of vertebral segments inwhich particular segments appeared to have a morphology typical of moreanterior segments. These transformations, which are summarized in FIG.12, were evident throughout the axial skeleton extending from thecervical region to the caudal region. Except for the defects seen in theaxial skeleton, the rest of the skeleton, such as the cranium and limbbones, appeared normal.

Anterior transformations of the vertebrae in mutant newborn animals weremost readily apparent in the thoracic region, where there was a dramaticincrease in the number of thoracic (T) segments. All wild-type miceexamined showed the typical pattern of 13 thoracic vertebrae each withits associated pair of ribs (FIGS. 11B and 11E). In contrast, homozygousmutant mice showed a striking increase in the number of thoracicvertebrae. All homozygous mutants examined had 4 to 5 extra pairs ofribs for a total of 17 to 18 (FIGS. 11D and 11G) although in over ⅓ ofthese animals, the 18th rib appeared to be rudimentary. Hence, segmentsthat would normally correspond to lumbar (L) segments L1 to L4 or L5appeared to have been transformed into thoracic segments in mutantanimals.

Moreover, transformations within the thoracic region in which onethoracic vertebra had a morphology characteristic of another thoracicvertebra were also evident. For example, in wild-type mice, the first 7pairs of ribs attach to the sternum, and the remaining 6 are unattachedor free (FIGS. 11E and 11H)). In homozygous mutants, there was anincrease in the number of both attached and free pairs of ribs to 10-11and 7-8, respectively (FIGS. 11G and 11J). Therefore, thoracic segmentsT8, T9, T10, and in some cases even T11, which all have free ribs inwild-type animals, were transformed in mutant animals to have acharacteristic typical of more anterior thoracic segments, namely, thepresence of ribs attached to the sternum. Consistent with this finding,the transitional spinous process and transitional articular processeswhich are normally found on T10 in wild-type animals were instead foundon T13 in homozygous mutants (data not shown). Additionaltransformations within the thoracic region were also noted in certainmutant animals. For example, in wild-type mice, the ribs derived from T1normally touch the top of the sternum. However, in 2/23 hybrid and ⅔129/SvJ homozygous mutant mice examined, T2 appeared to have beentransformed to have a morphology resembling that of T1; that is, inthese animals, the ribs derived from T2 extended to touch the top of thesternum. In these cases, the ribs derived from T1 appeared to fuse tothe second pair of ribs. Finally, in 82% of homozygous mutants, the longspinous process normally present on T2 was shifted to the position ofT3. In certain other homozygous mutants, asymmetric fusion of a pair ofvertebrosternal ribs was seen at other thoracic levels.

The anterior transformations were not restricted to the thoracic region.The anterior most transformation that we observed was at the level ofthe 6th cervical vertebra (C6). In wild-type mice, C6 is readilyidentifiable by the presence of two anterior tuberculi on the ventralside. In several homozygous mutant mice, although one of these twoanterior tuberculi was present on C6, the other was present at theposition of C7 instead. Hence, in these mice, C7 appeared to have beenpartially transformed to have a morphology resembling that of C6. Oneother homozygous mutant had 2 anterior tuberculi on C7 but retained oneon C6 for a complete C7 to C6 transformation but a partial C6 to C5transformation.

Transformations of the axial skeleton also extended into the lumbarregion. Whereas wild-type animals normally have only 6 lumbar vertebrae,homozygous mutants had 8-9. At least 6 of the lumbar vertebrae in themutants must have derived from segments that would normally have givenrise to sacral and caudal vertebrae as the data described above suggestthat 4 to 5 lumbar segments were transformed into thoracic segments.Hence, homozygous mutant mice had a total of 33-34 presacral vertebraecompared to 26 presacral vertebrae normally present in wild-type mice.The most common presacral vertebral patterns were C7/T18/L8 andC7/T18/L9 for mutant mice compared to C7/T13/L6 for wild-type mice. Thepresence of additional presacral vertebrae in mutant animals was obviouseven without detailed examination of the skeletons as the position ofthe hind-limbs relative to the forelimbs was displaced posteriorly by7-8 segments.

Although the sacral and caudal vertebrae were also affected inhomozygous mutant mice, the exact nature of each transformation was notas readily identifiable. In wild-type mice, sacral segments S1 and S2typically have broad transverse processes compared to S3 and S4. In themutants, there did not appear to be an identifiable S1 or S2 vertebra.Instead, mutant animals had several vertebrae that appeared to havemorphology similar to S3. In addition, the transverse processes of all 4sacral vertebrae are normally fused to each other although in newbornsoften only fusions of the first 3 vertebrae are seen. In homozygousmutants, however, the transverse processes of the sacral vertebrae wereusually unfused. In the caudal-most region, all mutant animals also hadseverely malformed vertebrae with extensive fusions of cartilage.Although the severity of the fusions made it difficult to count thetotal number of vertebrae in the caudal region, we were able to count upto 15 transverse processes in several animals. We were unable todetermine whether these represented sacral or caudal vertebrae in themutants because we could not establish morphologic criteria fordistinguishing S4 from caudal vertebrae even in wild-type newbornanimals. Regardless of their identities, the total number of vertebraein this region was significantly reduced from the normal number ofapproximately 30. Hence, although the mutants had significantly morethoracic and lumber vertebrae than wild-type mice, the total number ofsegments was reduced in the mutants due to the truncation of the tails.

Heterozygous mice also showed abnormalities in the axial skeletonalthough the phenotype was much milder than in homozygous mice. The mostobvious abnormality in heterozygous mice was the presence of anadditional thoracic segment with an associated pair of ribs (FIGS. 11Cand 11F). This transformation was present in every heterozygous animalexamined, and in every case, the additional pair of ribs was attached tothe sternum (FIG. 11I). Hence, T8, whose associated rib normally doesnot touch the sternum, appeared to have been transformed to a morphologycharacteristic of a more anterior thoracic vertebra, and L1 appeared tohave been transformed to a morphology characteristic of a posteriorthoracic vertebra. Other abnormalities indicative of anteriortransformations were also seen to varying degrees in heterozygous mice.These included a shift of the long spinous process characteristic of T2by one segment to T3, a shift of the articular and spinous processesfrom T10 to T11, a shift of the anterior tuberculus on C6 to C7, andtransformation of T2 to T1 where the rib associated with T2 touched thetop of the sternum.

In order to understand the basis for the abnormalities in axialpatterning seen in GDF-11 mutant mice, we examined mutant embryosisolated at various stages of development and compared them to wild-typeembryos. By gross morphological examination, homozygous mutant embryosisolated up to day 9.5 of gestation were not readily distinguishablefrom corresponding wild-type embryos. In particular, the number ofsomites present at any given developmental age was identical betweenmutant and wild-type embryos, suggesting that the rate of somiteformation was unaltered in the mutants. By day 10.5-11.5 p.c., mutantembryos could be easily distinguished from wild-type embryos by theposterior displacement of the hind-limb by 7-8 somites. Theabnormalities in tail development were also readily apparent at thisstage. Taken together, these data suggest that the abnormalitiesobserved in the mutant skeletons represented true transformations ofsegment identities rather than the insertion of additional segments, forexample, by an enhanced rate of somitogenesis.

Alterations in expression of homeobox containing genes are known tocause transformations in Drosophila and in vertebrates. To see if theexpression patterns of Hox genes (the vertebrate homeobox containinggenes) were altered in GDF-11 null mutants we determined the expressionpattern of 3 representative Hox genes, Hoxc-6, Hoxc-8 and Hoxc-11, inday 12.5 p.c. wild-type, heterozygous and homozygous mutant embryos bywhole mount in situ hybridization. The expression pattern of Hoxc-6 inwild-type embryos spanned prevertebrae 8-15 which correspond to thoracicsegments T1-T8. In homozygous mutants, however, the Hoxc-6 expressionpattern was shifted posteriorly and expanded to prevertebrae 9-18 (T2-T11). A similar shift was seen with the Hoxc-8 probe. In wild-typeembryos, Hoxc-8 was expressed in prevertebrae 13-18 (T6-T11) but, inhomozygous mutant embryos, Hoxc-8 was expressed in prevertebrae 14-22(T7-T15). Finally, HoxC-11 expression was also shifted posteriorly inthat the anterior boundary of expression changed from prevertebrae 28tin wild-type embryos to prevertebrae 36 in mutant embryos. (Note thatbecause the position of the hind-limb is also shifted posteriorly inmutant embryos, the Hoxc-11 expression patterns in wild-type and mutantappeared similar relative to the hind-limbs). These data provide furtherevidence that the skeletal abnormalities seen in mutant animalsrepresent homeotic transformations.

The phenotype of GDF-11 mice suggested that GDF-11 acts early duringembryogenesis as a global regulator of axial patterning. To begin toexamine the mechanism by which GDF-11 exerts its effects, we determinedthe expression pattern of GDF-11 in early mouse embryos by whole mountin situ hybridization. At these stages the primary sites of GDF-11expression correlated precisely with the known sites at which mesodermalcells are generated. Expression of GDF-11 was first detected at day8.25-8.5 p.c. (8-10 somites) in the primitive streak region, which isthe site at which ingressing cells form the mesoderm of the developingembryo. Expression was maintained in the primitive streak at day 8.75,but by day 9.5 p.c., when the tail bud replaces the primitive streak asthe source of new mesodermal cells, expression of GDF-11 shifted to thetail bud. Hence at these early stages, GDF-11 appears to be synthesizedin the region of the developing embryo where new mesodermal cells ariseand presumably acquire their positional identity.

The phenotype of GDF-11 knockout mice in several respects resembles thephenotype of mice carrying a deletion of a receptor for some members ofthe TGF-β superfamily, the activin type IIB receptor (ActRIIB). As inthe case of GDF-11 knockout mice, the ActRIIB knockout mice have extrapairs of ribs and a spectrum of kidney defects ranging from hypoplastickidneys to complete absence of kidneys. The similarity in the phenotypesof these mice raises the possibility that ActRIIB may be a receptor forGDF-11. However, ActRIIB cannot be the sole receptor for GDF-11 becausethe phenotype of GDF-11 knockout mice is more severe than the phenotypeof ActRIIB mice. For example, whereas the GDF-11 knockout animals have4-5 extra pairs of ribs and show homeotic transformations throughout theaxial skeleton, the ActRIIB knockout animals have only 3 extra pairs ofribs and do not show transformations at other axial levels. In addition,the data indicate that the kidney defects in the GDF-11 knockout miceare also more severe than those in ActRIIB knockout mice. The ActRIIBknockout mice show defects in left/right axis formation, such as lungisomerism and a range of heart defects that we have not yet observed inGDF-11 knockout mice. ActRIIB can bind the activins and certain BMPs,although none of the knockout mice generated for these ligands showdefects in left/right axis formation.

If GDF-11 does act directly on mesodermal cells to establish positionalidentity, the data presented here would be consistent with either shortrange or morphogen models for GDF-11 action. That is, GDF-11 may act onmesodermal precursors to establish patterns of Hox gene expression asthese cells are being generated at the site of GDF-11 expression, oralternatively, GDF-11 produced at the posterior end of the embryo maydiff-use to form a morphogen gradient. Whatever the mechanism of actionof GDF-11 may be, the fact that gross anterior/posterior patterningstill does occur in GDF-11 knockout animals suggests that GDF-11 may notbe the sole regulator of anterior/posterior specification. Nevertheless,it is clear that GDF-11 plays an important role as a global regulator ofaxial patterning and that further study of this molecule will lead toimportant new insights into how positional identity along theanterior/posterior axis is established in the vertebrate embryo.

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1. A method for identifying a compound that affects GDF-11 activity orgene expression, comprising: a) incubating the compound with a humanGDF-11 polypeptide as set forth in SEQ ID NO: 2, or with a recombinantcell expressing a human GDF-11 as set forth in SEQ ID NO: 2 underconditions sufficient to allow the components to interact; and b)determining the effect of the compound on GDF-11 activity or expression.2. The method of claim 1, wherein the effect is inhibition of GDF-11activity or expression.
 3. The method of claim 2, wherein the effect isdetermined by comparing expression of GDF-11 prior to incubating withthe compound with the expression of GDF-11 after incubating with thecompound.
 4. The method of claim 2, wherein the effect is measured byNorthern Blot analysis.
 5. The method of claim 2, wherein the effect ismeasured by Western Blot analysis.
 6. The method of claim 2, wherein theeffect is measured by muscle fiber analysis.
 7. The method of claim 2,wherein the components interact in solution.
 8. The method of claim 2,wherein the components interact in solid phase.
 9. The method of claim2, wherein the components interact in a cell.
 10. The method of claim 2,wherein the compound is a member of a combinatorial library.